Exploring ParE Toxin Dynamics: from Impact to Quantification in Bacterial Cells

Abstract

The increasing prevalence of antimicrobial resistance (AMR) in pathogenic bacteria has brought significant challenges to global public health. However, it has been decades since the discovery of the last antibiotic, making it essential to explore novel therapeutic targets and mechanisms to combat bacterial infections. Bacterial toxin-antitoxin (TA) systems, which exist widely among bacterial species, have been attracting attention in the field for their potential to be co-opted for health purposes. These non-secreted TA systems typically consist of a stable toxin that inhibits essential cellular processes, leading to cell growth inhibition or cell death, and a labile antitoxin (RNA or protein) that neutralizes the cognate toxin under normal growth conditions. In the case of Type-II ParDE TA system, the ParE toxin protein is neutralized by the ParD antitoxin protein through direct protein-protein interactions. Under stress conditions, degradation of the ParD antitoxin releases the ParE toxin. Similar to quinolone antibiotics, this liberated ParE toxin interacts with and inhibits DNA gyrase, causing the accumulation of double-strand breaks (DSBs) in DNA in the bacterial cell, which could lead to cell death. The detrimental DNA gyrase inhibition mediated by ParE toxins and their widespread presence in Gram-negative bacteria of concern make them a potential, potent agent for antibacterial drug development. The idea of co-opting TA systems as a strategic tool to control bacterial growth is still in its early stage due to the lack of knowledge on how to artificially activate the toxins in vivo. This dissertation focuses on ParE subfamily members from Pseudomonas aeruginosa and Mycobacterium tuberculosis and aims to provide new insights for the proof of concept of co-opting the ParE toxins as a novel therapeutic agent. By exploring the effects of ParE toxin-mediated DNA damage on bacterial growth, genetic resistance, and antibiotic susceptibility, this work addresses a key gap in our understanding of the role of the ParE toxin in bacterial physiology and therapeutic development. This dissertation comprises three key research chapters: The work in Chapter II investigated the phenotypic impacts of induced ParE toxin expression on bacterial growth, genetic mutation, and antibiotic susceptibility. The expression of ParE toxins—originating from P. aeruginosa and M. tuberculosis—was induced in P. aeruginosa cells and Escherichia coli surrogate cells. Differential toxicity profiles were noted, with the PaParE1 toxin exhibiting essentially no toxicity and the other ParE toxins exhibiting dose-dependent toxicity. ParE toxin-mediated DSBs in DNA trigger error-prone DNA repair pathways, such as the SOS response, which could lead to the accumulation of genetic mutations potentially contributing to the emergence of antibiotic resistance. Results indicated that the expression of potent ParE toxins led to an increased mutation frequency, except for the case of the attenuated ParE toxin. However, this increase in mutation frequency did not translate into significant resistance against a broad spectrum of common clinical antibiotics within the observation period. These findings support the concept of co-opting TA systems as an antibacterial approach. The work in Chapter III uncovered a survival mechanism that E. coli cells use to evade the lethal effects of plasmid-mediated ParE toxin expression. In the work of Chapter II, we observed an interesting phenotype where E. coli cells, after exposure to the plasmid-mediated inducible expression of the ParE1 toxin from M. tuberculosis, became “insensitive” to subsequent induction of the ParE1 toxin. Moreover, the proportion of these insensitive cells increased with continued passages in the presence of the inducer. This phenotype was not correlated with changes in the plasmid sequence and could not be rescued by increasing the inducer uptake. Instead, it was associated with a marked reduction in plasmid copy number (PCN). This reduction in PCN was reproducible across various E. coli strains and ParE toxins, indicating a generalized response mechanism. Furthermore, bacterial whole genome sequencing revealed a N845S residue substitution in DNA polymerase I, which is known to participate in the replication of the type of plasmid used in our experiments. This observed survival strategy of reducing PCN highlights the adaptability of bacterial cells to stress conditions and provides valuable insights into microbial adaptation and genetic engineering methods. The work in Chapter IV validated the feasibility of using a novel bio-layer interferometry (BLI)-based method to quantify low-abundance ParE protein molecules in cell lysate. In the work of Chapter II, we noticed that despite sharing a conserved three-dimensional structure, the ParE toxins exhibited varying toxicity profiles. This differential potency may stem from variations in protein sequence or expression levels. Traditional detection methods like Western blot and mass spectrometry failed to detect those potent ParE toxins due to their toxic nature and resulting low abundance in cell lysate. To overcome the limitation, we employed the highly sensitive BLI technique. Using the attenuated ParE1 toxin from P. aeruginosa, which allows for robust expression and purification, we optimized the specific binding of ParE1 toxin molecules to biosensors by adjusting the number of dips and cell lysate concentration in the running buffer. We established linear relationships between the specific binding signals and ParE1 toxin concentrations using different types of biosensors, demonstrating the feasibility of using BLI-based method for the quantification of ParE protein molecules in cell lysate. Overall, this dissertation not only provides a comprehensive view on the phenotype impacts of ParE toxins on bacterial growth, genetic mutation, DNA stability, and antibacterial response, laying the foundation for co-opting TA systems as an antibacterial strategy, but also introduces a novel methodological approach. This approach will enhance our understanding of the molecular dynamics of toxin proteins, which facilitates future studies on TA system biology.